Bis [o-(14-benzoylaconine-8-yl)] esters

ABSTRACT

The present invention concerns novel compounds having the structure (I). Another object of the invention is the use of compounds (I) for the fabrication of a medicament which is used for the treatment of tumour diseases.

The present invention relates to novel compounds which are alkaloidsrelated to [O-(14-benzoylaconine-8-yl)] moiety.

The invention furthermore concerns a method for producing thesecompounds, medicaments containing these, and their use in the treatmentof cancer.

Despite the wide variety of currently available anticancer drugs, theresearch and identification of new natural substances extracted fromplants with antiproliferative activity remains a priority. Only a smallnumber of available plants have been studied in this respect, andnatural biodiversity offers an unlimited field for the discovery ofpotential anticancer drugs.

In addition, it is to be noticed that numerous plant extracts havecurrently been used in traditional medicine and their activeconstituents have never been isolated and identified. Amongst new drugs,natural products and hemisynthetic derivatives of alkaloids have beenreported to possess antiproliferative activity.

One example of these natural products is a traditional medicinal plantgrowing in Kirghiz Republic, which has been preliminary screened. Moreprecisely, this plant belongs to the genus Aconitum, familyRanunculaceae and it has been selected for detailed investigationbecause of its traditional use against cancer in this country for manyyears. Indeed, the roots of Aconitum karakolicum have been used againstcancer in some prescriptions of traditional kirghiz medecine.

Aconitum karakolicum Rapcs is an herbaceous perennial plant with a tallleafy stem (70-130 cm) bearing violet or blue zygomorphous flowers onlong, packed racemes. The underground part of the plant is representedby cone-shaped tubercles measuring 2.0−2.5×0.7-1.0 cm, which grow bysticking to each other side by side. Aconitum karakolicum is endemic toCentral Asia.

All the parts of Aconitum species contain alkaloids of the diterpenoidgroup. The maximum content in alkaloids has been found in tuberclesafter the period of vegetation (August-October).

The principal alkaloids identified in this plant are:

-   -   aconitine (0.8-1%), which is well-known to have a strong        toxicity and to exhibit both centrally and peripherally effects        through preventing the normal closure of sodium channels,    -   karakoline (0.05%),    -   karakolidine (0.05%),    -   zongorine (0.1%), and    -   zongoramine (0.01%).

Napelline, aconifine, acetylnapelline and karakonitine have also beenidentified.

But, it has been found that none of these compounds bear anyantiproliferative activity, whereas plant extracts do.

Thus, a work of isolating and identifying the compounds present inextracts of the A. Karakolicum roots, which were responsible for thisactivity, has been undertaken and has been detailed in the publicationof A. Chodoeva et al., entitled “8-O-Azeloyl-14-benzoylaconine: A newalkaloid from the roots of Aconitum karakolicum Rapcs and itsproliferative activities”; Bioorg. Med. Chem. (2005), 13, 6493-6501.

Indeed, this publication discloses a novel compound:8-O-azeloyl-14-benzoylaconine, which has the following structure (1):

8-O-azeloyl-14-benzoylaconine had never been described in the chemicalliterature, although several fatty acyl esters of aconine had beenpreviously described in Wie, X.; Xie, H.; Liu, M.; Ge, X., Heterocycles(2000), 53, 2027.

Aconine, which is itself devoid of antiproliferative activity, has thefollowing structure (2):

Aconitine, which has been above mentioned, is an aconine derivative andhas the following structure (3):

The 8-O-azeloyl-14-benzoylaconine of general formula O₄₁H₅₉NO₁₃ is anaconitine derivative, because it results in fact from the replacement ofthe acetyl group by an azelaidic acyl moiety on carbon 8 of theaconitine skeleton.

A feature of the compound of 8-O-azeloyl-14-benzoylaconine lies on itszwitterionic structure between the negative charge of the carboxylatefunction at the extremity of the azelaic acid chain and the positivecharge of the quaternary ammonium formed on the nitrogen atom of theheterocycle. Given the length of the azelaic chain, an internal ionicbond between these two moieties could be postulated.

Concerning azelaic acid, it has been shown to be an inhibitor ofmitochondrial oxidoreductases in tumour cells (Picardo, M.; Passi, S.;Sirlanni, M. C.; Fiorilli, M.; Russo, G. D.; Cortesi, E.; Barile, G.;Breathnach, A. S.; NazzaroPorro, M.; Biochem. Pharmacol. (1985), 34,1653).

As a consequence, azelaic acid was proposed as a general antitumouragent (Breathnach, A. S., Med. Hypotheses, (1999), 52, 221).

Azelaic acid has never been tested in the in vitro screening panel ofthe NCl, but some experiments have been performed on mouse tumours invivo, which concluded in the total absence of antitumour activity.

The antiproliferative activity observed for8-O-azeloyl-14-benzoylaconine cannot therefore be attributed to theactivity of its individual constituents.

However, according to the above mentioned publication A. Chodoeva etal., 8-O-azeloyl-14-benzoylaconine has been found responsible for theantiproliferative in vitro activity against three lines of human tumourcells in culture, which were:

-   -   HTC-15 (colon cancer),    -   A549 (lung cancer),    -   MCF-7 (breast cancer).

Its IC₅₀ was about 10-18 μM in these three cell lines, which is in linewith the activity of major anticancer drugs belonging to several classes(i.e. antimetabolites, alkylating agents, platinum compounds,topoisomerase inhibitors).

As above mentioned, it is intensively searched for novel compoundsexhibiting antiproliferative activity against human tumour cells.

Thus, it was therefore an object of the present invention to improve thedesign 8-O-azeloyl-14-benzoylaconine, which had revealed veryinteresting and promising properties against cancer and to provide withnovel compounds useful in the cancer chemotherapy.

According to the invention, this object is first of all solved byproviding new compounds useful in the cancer chemotherapy, which arealkaloids related to [O-(14-benzoylaconine-8-yl)] moiety.

More precisely, these novel compounds arebis[O-(14-benzoylaconine-8-yl)]esters.

Thus, according to the invention, novel compounds having the followingthe general structure (I) are provided:

wherein n is an integer comprised between 0 and 10, preferably between 3and 10, more preferably between 4 and 8.

Thereby, preferred compounds according to the invention are compoundswherein:

-   -   n is 5, so the compound        bis[O-(14-benzoylaconine-8-yl)]-pimelate, (i.e. PDD)    -   n is 6, so the compound bis[O-(14-benzoylaconine-8-yl)]-suberate        (i.e. SDD),    -   n is 7, so the bis[O-(14-benzoylaconine-8-yl)]azelate (i.e.        ADD).

Thus, bis[O-(14-benzoylaconine-8-yl)]-pimelate has the followingstructure:

Thus, bis[O-(14-benzoylaconine-8-yl)]-suberate (i.e. SDD) has thefollowing structure:

Thus, bis[O-(14-benzoylaconine-8-yl)]-azelate (i.e. ADD) has thefollowing structure:

Another object of the present invention relates to a method forproducing a compound according to the present invention.

The compounds according to the present invention are obtained bytransesterification carried out on the 8-acetyl functionality beared onthe aconitine moiety.

The reaction from aconitine is achieved at controlled reaction times andtemperature such that it avoids the formation of products of degradationand it optimizes the said reaction of transesterification.

It leads to corresponding mono-[O-(14-benzoylaconine-8-yl)] andbis-[O-(14-benzoylaconine-8-yl)]esters. So, a step of purification isnecessary in order to isolate the bis-[O-(14-benzoylaconine-8-yl)]estersaccording to the present invention.

More precisely, the method for the production of a compound according tothe present invention comprises the following steps:

a) stirring aconitine with a dicarboxylic acid at a temperature equal orlower than 80° C. in a solvent such DMF,

b) agitating the obtained mixture at room temperature such that thereaction of transesterification is carried out,

c) evaporating the solvent under reduced pressure,

d) purification of the oily residue obtained at the end of the step c)to isolate the compound according to the present invention.

In preferred embodiments of the invention, the dicarboxylic acid may beselected among the dicarboxylic acids which have a number of carbonatoms comprised between 2 and 12, preferably between 5 and 12, morepreferably between 6 and 10.

Most preferably, the dicarboxylic acid may be selected in the groupconsisting of pimelic acid or suberic acid or azelaic acid.

The stirring of the step a) may be carried out at a temperaturecomprised between 70° C. and 80° C., and during a time sufficient tocarry out the reaction, most preferably during 7 hours.

In a preferred embodiment of the present invention, the agitating of thestep b) is carried out during 16 hours in order to optimize thereaction.

More preferably, the evaporating of the solvent of the step c) iscarried out at a temperature lower than 70° C.

In a preferred embodiment of the present invention, the purification ofthe step d) is carried out by a semi-preparative high pressure liquidchromatography.

The conditions of said semi-preparative high pressure liquidchromatography may be the following:

-   -   Solvent A: trifluoroacetic acid/water; in proportion of 1/1000;    -   Solvent B: trifluoroacetic acid/acetonitrile, in proportion of        1/1000.

The elution conditions may consist in an injection of a volume of 0.5mL; gradient from 25% of B to 100% in 30 min.

The absorbance is monitored at 234 nm.

Another object of the present invention is a pharmaceutical compositionwhich comprises a compound according to the present invention.

The pharmaceutical composition may further comprise a suitable,pharmaceutically acceptable diluent or carrier substance.

In accordance with the invention, the above mentioned pharmaceuticalcomposition can be present in the form of tablets, capsules, droplets,and suppositories, preparations for injection or infusion for a peroral,rectal or parenteral use. Such administration forms and their productsare known to the person of skill.

Furthermore, the present invention concerns the use of a compoundaccording to the present invention for the fabrication of a medicament.

More precisely, the present invention concerns the use of a compoundaccording to the invention in which the medicament is used for thetreatment of tumour diseases. The tumour diseases may be colon or lungor breast cancer.

The following examples further illustrate the present invention. But, ofcourse, should not be construed as in any way limiting its scope.

First at all, the conditions of different techniques are detailed, whichare:

-   -   HPLC,    -   mass spectroscopy, and    -   NMR.        For all of the Examples the Conditions Used for the HPLC        Purification were the Following:

Column used: Ultrasep ES 10, RP 186.0 μm reversed phase, C18 column,250×10 mm (Bischoff, Germany).

Solvents Used:

-   -   A: trifluoroacetic acid/water; in proportion of 1/1000;    -   B: trifluoroacetic acid/acetonitrile, in proportion of 1/1000;

Elution conditions: injection of a volume of 0.5 mL; gradient from 25%of B to 100% in 30 min.

Absorbance was monitored at 234 nm.

For all of the Examples the Conditions of Mass Spectroscopy were theFollowing:

High resolution ESI mass measurements were performed on a AppliedBiosystems QStarmass spectrometer equipped with an electrospray sourcein positive mode.

The electrosprayneedle was maintained at 5000 V and operated at roomtemperature.

Samples were introduced by injection through a 10 μl_sample loop into a200 μL/min flow of methanol from the LC pump.

MS² analyses were performed with a collision energy ranging from 30 to50 and CAD collision gas ranging from 5 to 10.

For all of the Examples the Conditions of Nuclear Magnetic Resonance(NMR) were the Following:

The 1D and 2D NMR experiments were performed on a Bruker DPX400spectrometer at 400.13 and 100.6 MHz for ¹H and ¹³C experiments,respectively, equipped with a 5 mm broadband probe and Bo gradients. Allspectra were recorded using 9 mg of substance dissolved in 0.7 mL ofCDCl₃. Chemical shifts in ppm are given relative to TMS. The ¹H-¹H shiftcorrelated two dimensional (COSY) spectra are obtained using the COSY 90pulse sequence. Reference is made to Picardo, M.; Passi, S.; Sirlanni,M. C.; Fiorilli, M.; Russo, G. D.; Cortesi, E.; Barile, G.; Breathnach,A. S.; Nazzaro-Porro, M., Biochem. Pharmacol. (1985), 34, 1653.

The type of carbon is defined by 2 experiments 1D (DEPT 90 and 135). Theone-bond ¹H-¹³C chemical shift correlation (HMQC) spectra had beenobtained according to the Bax sequence using Bo gradient pulses for theselection of ¹H coupled to ¹³C carbons. Reference is made to Breathnach,A. S. Med. Hypoth. (1999), 52, 221.

The ¹H detected heteronuclear multiple bond correlation (HMBC) spectrawere recorded using the pulse sequence proposed by Bax and Summersinvolving a low pass Jn filter (3.8 ms) and a delay to observe thelong-range coupling (60 ms) as in the HMQC experiment, B_(o) gradientpulses were applied to select ¹H coupled to ¹³C nuclei. Gradientselected NOESY spectra were acquired with the NOESYGPPH Bruker pulseprogram.

Hereafter, the synthesis and the features of three compounds accordingto the present invention are detailed.

A) bis[O-(14-benzoylaconine-8-yl)]-pimelate (PDD) Synthesis of theCompound:

Aconitine (200 mg, 0.30 mmol) and pimelic acid (24 mg, 0.15 mmol) in DMF(5 ml) was stirred up during 7 hours at 80° C. The mixture is agitatedduring a night at room temperature. The solvent is then evaporated underreduced pressure at a temperature lower than 70° C. The oily residue ispurified by semi-preparative high pressure liquid chromatography.

NMR Features of the Obtained Compound:

White powder: ¹H NMR spectrum (400.13 MHz, CDCl₃) showed δ: 8.1 (d, 4H,J=7.6, H-2′/H-6′), 7.71 (t, 2H, J=7.4, H-4′), 7.58 (t, 4H, J=7.6,H-3′/H-5′), 5.04 (d, 2H, J=4.4, H-14), 4.63 (d, 2H, J=5.2, H-15), 4.44(br s, 2H, H-3), 4.27 (br d, 2H, H-6), 3.92 (m^(a), 2H, H-19b), 3.95 (s,6H, 16-OCH₃), 3.67 (s, 2H, H-18b^(a)), 3.64 (m, 2H, H-1), 3.53 (s, 6H,1-OCH₃), 3.48 (s, 2H, H-17), 3.47 (s, 6H, 18-OCH₃), 3.41 (s, 2H, 18e),3.39 (br d, 2H, H-16), 3.36 (s, 6H, 6-OCH₃), 3.31 (m^(a), 2H, H-19a),3.33 (m^(a), 4H, N—CH₂—CH₃), 3.08 (m^(a), 2H, H-9), 3.06 (s, 2H, H-5),2.74 (br d, 2H, H-7), 2.58 (br s, 2H, H-2b), 2.51 (br d, 2H, H-12b),2.49 (m^(a), 2H, H-10), 2.07 (d, 2H, J=10, H-12a), 1.87 (m, 2H,8-CO₂—(CH₂)1″b, 8-CO₂—(CH₂)₆″b), 1.60 (m, 10H, 8-CO₂—(CH₂)1″a,8-CO₂—(CH₂)₅″a, N—CH₂—CH₃, H-2aa), 1.12 (m, 2H, 8-CO₂—(CH₂)₂″b,8-CO₂—(CH₂)₄″b), 0.95 (m, 2H, 8-CO₂—(CH₂)₂″a, 8-CO₂—(CH₂)₄″a), 0.72(quint, 2H, J=7.2, 8-CO₂—(CH₂)₃″). ¹³C NMR (CDCl₃) δ: 174.9 (8-COO,8-COO″), 165.76 (C-7′), 133.8 (C-4′), 129.9 (C-2′/C-6′), 129.5 (C-1′),129 (C3′/C-5′), 90 (C-16), 89.98 (C-8), 82.48 (C-6), 79.83 (C-1), 78.12(C-15), 78.45 (C-14), 75.79 (C-18), 74.02 (C-13), 69.9 (C-3), 63.12(C-17), 62.01 (16-OCH₃), 59.3 (18-OCH₃), 59.05 (6-OCH₃), 55.36 (1-OCH₃),50.63 (C-19), 50.42 (C-11), 50.38 (N—CH₂—CH₃), 45.0 (C-5), 43.42 (C-4),43.47 (C-9), 41.91 (C-7), 40.09 (C-10), 35.01 (C-12), 34.27 (C-1″,C-5″), 29.49 (C-2″, C-4″), 28.02 (C-2), 23.86 (C-3″), 10; 99(N—CH₂—CH₃).

B) bis[O-(14-benzoylaconine-8-yl)]-suberate (SDD) Synthesis of theCompound:

Aconitine (200 mg, 0.30 mmol) and suberic acid (26 mg, 0.15 mmol) in DMF(5 mL) was stirred up during 7 hours at 80° C. The mixture is agitatedduring a night at room temperature. The solvent is then evaporated underreduced pressure at a temperature lower than 70° C. The oily residue ispurified by semi-preparative high pressure liquid chromatography.

Features of the Obtained Compound:

White powder: ¹H NMR spectrum (400.13 MHz, CDCl₃) showed δ: 8.00 (d, 4H,J=7.8, H-2′/H-6′), 7.58 (t, 2H, J=7.32, H-4′), 7.46 (t, 4H, J=7.66,H-3′/H-5′), 5.04 (d, 2H, J=4.9, H-14), 4.64 (d, 2H, J=5.36, H-15), 4.43(br s, 2H, H-3), 4.28 (d, 2H, J=6.1, H-6), 3.97 (d, 2H, J=12.96, H-19b),3.94 (s, 6H, 16-OCH₃), 3.67 (s, 2H, H-18ba), 3.64 (m, 2H, H-1), 3.53 (s,6H, 1-OCH₃), 3.47 (s, 2H, H-17), 3.43 (s, 6H, 18-OCH₃), 3.41 (s, 2H,18aa), 3.39 (d, 2H, J=5.4, H-16), 3.36 (s, 6H, 6-OCH₃), 3.31 (d, 2H,J=12.96, H-19a), 3.30 (m^(a), 4H, N—CH₂—CH₃), 3.10 (m, 2H, H-9), 3.07(s, 2H, H-5), 2.74 (d, 2H, J=6.1, H-7), 2.58 (br s, 2H, H-2b), 2.51 (brd, 2H, H-12b), 2.49 (m, 2H, H-10), 2.07 (d, 2H, J=9.9, H-12a), 1.94 (m,2H, 8-CO2-(CH2)1″b, 8-CO2-(CH2)6″b), 1.63 (m, 2H, 8-CO2-(CH2)1″a,8-CO2-(CH2)6″a), 1.60 (t, 6H, J=6.98, N—CH₂—CH₃), 1.56 (br d, 2H,H-2aa), 1.24 (m, 2H, 8-CO2-(CH2)2″b, 8-CO2-(CH2)5″b), 1.09 (m, 2H,8-CO2-(CH2)2″a, 8-CO2-(CH2)5″a), 0.83 (m, 4H, 8-CO2-(CH2)3″,8-CO2-(CH2)4″). ¹³C NMR (CDCl3) δ: 175.1 (8-COO, 8-COO″), 165.8 (C-7′),133.8 (C-4′), 129.9 (C-2′/C-6′), 129.5 (C-1′), 129 (C3′/C-5′), 90(C-16), 89.97 (C-8), 82.51 (C-6), 79.82 (C-1), 78.82 (C-15), 78.40(C-14), 75.75 (C-18), 74.01 (C-13), 70 (C-3), 63.13 (C-17), 61.95(16-OCH₃), 59.26 (18-OCH₃), 59.01 (6-OCH₃), 55.29 (1-OCH₃), 50.64(C-19), 50.51 (C-11), 50.28 (N—CH₂—CH₃), 45.0 (C-5), 43.43 (C-4), 43.40(C-9), 41.87 (C-7), 40.06 (C-10), 35.0 (C-12), 34.49 (C-1″, C-6″), 29.50(C-2), 28.54 (C-3″, C-4″), 24.05 (C-2″, C-5″), 10; 94 (N—CH₂—CH₃).Furthermore, an ESI spectrum of bis[O-(14-benzoylaconine-8-yl)]-suberatehas been carried out. Two peaks are observed on this spectrum: a majorone at m/z 673.3 which corresponds to a doubly charged mass and a minorone at m/z 1345.7 which corresponds to a mono charged peak, thisspectrum enables us to confirm.High resolution mass measurements were also performed to confirm theformula of the synthesized compound. The experimental mass obtained onthe mono-charged peak is 1345.6829. The theoretical mass for the formulaC₇₂H₁₀₁N₂O₂₂ is 1345.6840 (high resolution mass).MS² experiments were also performed. These measurements were done withthe mono charged and the doubly charged peaks. The fragmentation of m/z1346.7 (mono charged peak) produced a major fragmentation peak at m/z760 which corresponds to the loss of one of the aconitine unit.Fragmentation of m/z 673 (doubly charged peak) produced more fragmentswhich correspond to the mechanism of fragmentation of the aconitineunit.

C) bis[O-(14-benzoylaconine-8-yl)]azelate (ADD) Synthesis of theCompound:

Aconitine (200 mg, 0.30 mmol) and azelaic acid (29 mg, 0.15 mmol) in DMF(5 mL) was stirred up during 7 hours at 80° C. The mixture is agitatedduring a night at room temperature. The solvent is then evaporated underreduced pressure at a temperature lower than 70° C. The oily residue ispurified by semi-preparative high pressure liquid chromatography.

Features of the Obtained Compound:

White powder: ¹H NMR spectrum (400.13 MHz, CDCL₃) showed δ: 8.03 (d, 4H,J=7.2, H-2′/H-6′), 7.56 (t, 2H, J=7.4, H-4′), 7.45 (t, 4H, J=7.2,H-3′/H-5′), 5.09 (d, 2H, J=5, H-14), 4.51 (d, 2H, J=5.2, H-15), 4.30 (brs, 2H, H-3), 4.12 (d, 2H, J=6.1, H-6), 4.06 (d, 2H, J=11.91, H-19b),3.79 (s, 6H, 16-OCH₃), 3.52 (s, 2H, H-18ba), 3.47 (m, 2H, H-1), 3.36 (s,6H, 1-OCH₃), 3.29 (s, 2H, H-17), 3.27 (s, 6H, 18-OCH₃), 3.24 (s, 2H,18e), 3.22 (br s, 2H, H-16), 3.19 (s, 6H, 6-OCH₃), 3.11 (d, 2H, J=11.9,H-19a), 3.16 (m, 4H, N—CH₂—CH₃), 2.90 (m, 2H, H-9), 2.86 (s, 2H, H-5),2.55 (d, 2H, J=6.1, H-7), 2.34 (br s, 2H, H-2b), 2.28 (br d, 2H, H-12b),2.24 (m, 2H, H-10), 2.02 (br d, 2H, H-12a), 1.82 (m, 2H, 8-CO₂—(CH₂)1″b,8-CO₂—(CH₂)7″b), 1.50 (m, 2H, 8-CO₂—(CH₂)1″a, 8-CO₂—(CH₂)7″a), 1.42 (t,6H, J=7.01, N—CH₂—CH₃), 1.35 (br s, 2H, H-2aa), 1.13 (m, 4H,8-CO₂—(CH₂)2″b, 8-CO₂—(CH₂)3″b, 8-CO₂—(CH₂)5″b, 8-CO₂—(CH₂)6″b), 1.04(m, 4H, 8-CO₂—(CH₂)2″a, 8-CO₂—(CH₂)3″a, 8-CO₂—(CH₂)5″a, 8-CO₂—(CH₂)6″a),0.87 (m, 2H, 8-CO₂—(CH₂)4″). ¹³C NMR (CDCl₃) δ: 175.1 (8-COO, 8-COO″),166 (C-7′), 134 (C-4′), 130 (C-2′/C-6′), 129.7 (C-1′), 129.3 (C3′/C-5′),90.3 (C-16), 90.1 (C-8), 82.9 (C-6), 80.1 (C-1), 79.0 (C-15), 78.7(C-14), 76.0 (C-18), 74.3 (C-13), 70.3 (C-3), 63.4 (C-17), 60.9(16-OCH₃), 59.5 (18-OCH₃), 59.06 (6-OCH₃), 55.6 (1-OCH₃), 50.7 (C-19),50.51 (C-11), 50.50 (N—CH₂—CH₃), 45.4 (C-5), 43.7 (C-4), 43.40 (C-9),41.9 (C-7), 40.5 (C-10), 35.3 (C-12), 34.49 (C-1″, C-7″), 28.8 (C-2″,C-6″), 26.54 (C-3″, C-5″), 24.05 (C-4″), 11 (N—CH₂—CH₃).

Evaluation of bis[O-(14-benzoylaconine-8-yl)]azelate (ADD)-suberate(SDD) and -pimelate (PDD) alkaloids In Vitro Cytotoxicity Test AgainstHuman Tumour Cell Lines Materials and Methods.

Human tumour cell lines A549 (lung cancer), HCT-15 (colon cancer) andMCF-7 (breast cancer) have been obtained from the DevelopmentalTherapeutics Program of the National Cancer Institute (Rockville, Md.,USA). Cells were routinely grown with RPMI 1640 medium supplemented with10% foetal calf serum, both obtained from Biochrom AG (Berlin, Germany).They were grown on Petri dishes (Nunc, Denmark) at 37° C. in ahumidified atmosphere containing 5% CO₂. Cells were replicated every 4days and the medium changed once in-between.Cytotoxicity has been evaluated on exponentially growing cells grown for24 h in presence of the compounds to be tested. Briefly, 4000 cells wereseeded in 96-well plates with 200 μL of complete medium; 24 hours laterthe medium was supplemented with a series of different concentrations ofcompounds and the contact maintained for 24 h. Compounds were dissolvedin pure water and sterilised using polycarbonate membrane filters of0.22 μm (Millipore, Molsheim, France). The cells were then allowed tore-grow for 48 hours; viable and metabolically active cells werequantitatively estimated by coloration with the MTT dye(1-(4,5-dimethylthyazol-2-yl)-3,5-diphenylformazan), Sigma-AldrichChimie, Saint-Quentin-Fallavier, France). The amount of substanceallowing a 50% decrease in cell numbers is indicative of thecytotoxicity and represents the IC₅₀ of compound.The following table 1 shows the cytotoxicity effects of the threebis[O-(14-benzoylaconine-8-yl)]-azelate (ADD),bis[O-(14-benzoylaconine-8-yl)]-suberate (SDD) andbis[O-(14-benzoylaconine-8-yl)]-pimelate (PDD).More precisely, table 1 summarizes the IC₅₀ values (μM) of:

-   bis[O-(14-benzoylaconine-8-yl)]-azelate (ADD),-   bis[O-(14-benzoylaconine-8-yl)]-suberate (SDD),-   bis[O-(14-benzoylaconine-8-yl)]-pimelate (PDD), and-   8-O-azeloyl-14-benzoylaconine (Comparison 1), and-   four major anticancer drugs (Comparison 2), as they appear in the    data base of the Development Therapeutic Program of the National    Cancer Institute, and which are:    -   fluorouracil (i.e. 5-FU),    -   cisplatin,    -   etoposide,    -   mephalan        tested on:    -   A549,    -   HCT-15, and    -   MCF-7 human tumour cell lines,

TABLE 1 IC₅₀ values (μM) of several compounds tested on A459, HCT-15 andMCF-7 human tumour cell lines The IC50 values of the substances, in μMA549 HCT-15 MCF-7 ADD 19.13 22 15.45 SDD 18.7 3.7 3.7 PDD 3.76 3.76 7.5Comparison 1 8-O-azeloyl-14- 16.8 19.4 10.3 benzoylaconine Comparison 25-FU 2.11 5.69 1.75 cisplatin 3.08 7.21 3.01 etoposide 3.61 17.6 5.73mephalan 28.7 39.4 11.1It can be observed from the table 1 that all the compounds according tothe invention show inhibitory effects. The best results were found forthe two SDD and PDD with values comparable with those of the four majoranticancer drugs (comparison 2). On the other hand, ADD showedinhibitory effect quite similar as those for corresponding8-O-azeloyl-14-benzoylaconine.Hereafter, studies, which were carried out on the most potentbis[O-(14-benzoylaconine-8-yl)]-suberate (SDD) of these three compoundsaccording to the invention, are described.

Partition Coefficients: log D (pH 7.4) of SDD

Partition coefficients between water/octanol allow evaluating thewater/lipo-solubility of the compound. This plays a major role indetermination of pharmacokinetic parameters such as penetration ofvarious biologic barriers and distribution within living organisms.

The relative log D (pH 7.4) in this study was assessed by the micro HPLCmethod which is disclosed in A microscale HPLC method for the evaluationof octanol-water partition coefficients in a series of new2-amino-2oxazolines Pehourcq, F.; Thomas, J.; Jarry, C., J. Liq.Chromatogr. Relat. Technol. (2000), 23, 443-453.

These determinations were performed with a chromatographic apparatus(Spectra Series, San Jose, USA). A reversed phase column was used: aStability RP18 (4.6×150 mm; 5 μm particle size) with a mobile phaseconsisting of acetonitrile (+1‰ trifluoroacetic acid)−water (+1‰trifluoroacetic acid) (40:60 v/v).

The compound was partitioned between n-octanol (HPLC grade) andphosphate buffer (pH=7.4). Octanol was presatured with buffer, andconversely. An amount of 1 mg of the compound was dissolved in anadequate volume of methanol in order to achieve 1 mg/mL stock solutions.Then, an appropriate aliquot of this methanolic solution was dissolvedin buffer to obtain final concentration of 100 μg/mL. Under the abovedescribed chromatographic conditions, 20 μL of this aqueous phase wasinjected into the chromatograph, leading to the determination of a peakarea before partitioning (W0).

In screw-capped tubes, 2000 μL of the aqueous phase (V_(ag)) was thenadded to 10 μL of n-octanol (V_(oct)) at pH=7.4. The mixture was shakenby mechanical rotation during 30 min. Centrifugation was achieved at3000 rpm in 15 min. An amount of 20 μL of the lower phase was injectedinto the chromatograph column. This led to the determination of a peakarea after partitioning (W₁). Log D was calculated from the formula: logD=log [(W₀−W₁)V_(aq)/W₁V_(oct)].

Concerning the compound SDD, a value of 2.86 of log D (pH 7.4) has beenfound, which corresponds to a relatively high liposolubility, favorableto a trans-membrane penetration of various biologic barriers anddistribution within living organisms, according to the followingpublication: Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P.J., Adv. Drug Delivery Rev. (1997), 23, 3-25.

Experimental and Computational approaches to estimate solubility andpermeability in drug discovery and development setting, in which anempirical scale of liposolubility is disclosed.

Effect of bis[O-(14-benzoylaconine-8-yl)]-suberate (SDD) on cell cyclearrest of different tumour cell lines Method.

10⁶ Cells of A549, HCT-15 and MCF-7 cell lines have been seeded in Petridishes with RPMI-1640 medium. 24 h later, SDD has been dissolved inwater at a concentration of 20 μM and was directly added to the culturemedium. Contact was maintained for 72 h. Then, cells have been detachedby trypsinization, collected and rinsed by PBS and counted onhemocytometer. Cells were centrifuged for 5 min at 1500 rpm, thenresuspended in a defined volume of PBS to obtain 2.5×10⁶ cell/mL. 100 μLof cell suspension was digested to nuclei by trypsin for 10 min. Then, asolution containing trypsin inhibitor and RNAse was added to cellsuspension and left for 10 min. Finally, cells were stained by propidiumiodide and were analysed by flow-cytometry apparatus. Distribution ofcells among the different phases of the cycle was determined by theprocedure disclosed in: A detergent-trypsin method for preparation ofnuclei for flow cytometric DNA analysis. Vindelov, L.; Christensen, I.;Nissen, N.; Cytometry (1983), 3, 323-327.The following table 2 summarizes the results obtained

-   -   G0G1: cell in the phase of GOG1    -   G2M: cells in the phase of G2M    -   S: cells in the phase of DNA synthesis    -   Sub G1: apoptotic cells    -   SUP G2M: diploidic cells

TABLE 2 Influence of SDD on tumour cell cycle arrest percentages oftotal cell population. Control Tumour cell (untreated lines cells) SDD(20 μM) A549 ALL % 100 100 G0G1 % 76.28 53.04 G2M % 9.15 16.42 S % 12.4225.49 SubG1 % 1.74 3.34 SUP G2M % 0.72 2.09 HCT-15 ALL % 100 100 G0G1 %69.83 30.67 G2M % 11.94 15.72 S % 15.19 26.35 SubG1 % 2.29 23.65 SUP G2M% 0.81 2.59 MCF-7 ALL % 100 100 G0G1 % 66.01 58.73 G2M % 13.51 16.96 S %16.56 21.44 SubG1 % 2.96 2.05 SUP G2M % 0.87 1.72The following table 3 summarizes the percentages of survival cells indifferent cell cycles.

TABLE 3 Percentage of survival cells in different cell cycles A549HCT-15 MCF-7 Control SDD Control SDD Control SDD G0G1 % 77.96 55.8672.01 42.16 68.7 60.46 G2M % 9.35 17.29 21.32 21.61 14.07 17.46 S % 12.726.84 15.67 36.23 17.23 22.08

Results

As the table 2 shows, SDD, at the dose of 20 μM, it led to a significantaccumulation of A549 and HCT-15 cells and moderate accumulation of MCF-7cells in the G2M and S phases, which is characteristic of cell cyclearrest at these stages of development. The numbers of apoptotic cells(Sub G1) was significantly increased in the test with HCT-15 cell lines.

It is important to notice that the number of cells in the G2M phaseincreased respectively by 85% and 75% in A549 and HCT-15 cell lines, andthe number of cells arrested at S phase increased by more than 100% inboth cell types as compared to control.

Upon SDD treatment similar trend of accumulation in G2M and S phase werenoticed in MCF-7 cells but to a lower degree. The effect on cell cyclewas not correlated with the cytotoxicity of SDD: A549 cell distributionin the cell cycle was altered at a SDD concentration close to IC₅₀,whereas MCF-7 cell distribution in the cycle was not altered at a SDDconcentration 5 times higher than IC₅₀. This makes us to supposemultiple action mechanisms, not only limited to inhibition of DNAsynthesis and block of G2M phase.

Study of the Effect of SDD on Topoisomerase I Cleavage Activity.

DNA topoisomerase 1 (i.e. Top 1) is an essential nuclear enzyme involvedin the regulation of DNA topology associated with most DNA transactions,including replication, transcription recombination and chromatinremodelling. Its role is to introduce transient single strand breaks induplex DNA via the formation of a covalent bond between the 3′-phosphateof the cleaved strand and enzyme. Within the covalent Top 1-DNA complex,rotation of the broken strand around the uncleaved strand results in DNAsupercoil relaxation. DNA continuity is then restored by Top 1 catalysedreligation of the 5′-hydroxyl termini. Top 1 poison, such asCamptothecin (CPT) stabilizes the Top 1-DNA complex, and consequently,inhibit DNA religation.

The influence of SDD has been studied at various concentrations on Top 1cleavage activity in comparison with Camptothecin.

The method consists in an incubation of 3′-labeled oligonucleotides withenzyme Top1, or Top 1+CPT or Top 1-SDD. Top 1 forms covalent bind withthe 3′-terminus of a DNA single strand beak and then religates DNA,which is revealed as a single band, Camptothecin, as a positive controlstabilize the complex Top1-DNA, inhibiting religation of DNA, thus, isrevealed as double band.

Method. Oligonucleotide Labelling

3′-end labelling of the scissile strand was performed as describedpreviously in the publication: Conversion of topoisomerase I cleavagecomplexes on the leading strand of ribosomal DNA into 5′-phosphorylatedDNA double-strand breaks by replication runoff. Pommier, Y.; Jenkins,J.; Kohlhagen, G.; and Leteurtre, F.; Mutat. Res. (1995), 337, 135-145.

Thus, 10 pmol of deoxynucleotidyltransferase was incubated in labellingbuffer (100 mM potassium cacodylate, pH 7.2, 2 mM CaCl₂, 200 μM DTT) for1 h at 37° C.

The reaction mixture was passed through a G-25 Sephadex spin column bycentrifugation at 1000 g for 5 min to remove the excess ofunincorporated nucleotide.

The 3′-labeled oligonucleotide was mixed with the same amount ofunlabeled complementary strand in annealing buffer (i.e. 10 mM Tris-HCl,pH 7.8, 100 mM Na₂EDTA) and annealed by heating the reaction mixture for5 min at 95° C. followed by slow cool down at room temperature.

Top1-catalysed Cleavage Assays

Top1-catalysed cleavage assays were performed using either full duplexoligonucleotides or partially double-stranded oligonucleotides, referredto as suicide substrates.

For each reaction, 20 fmol of 3′-labeled substrate at a concentration of10 μM was incubated with 0.2 μmol of purified human recombinant Top 1 ina buffer containing 10 mM Tris-HCl, pH 7.5, 50 mM KCl, 5 mM MgCl₂, 0.1mM Na₂EDTA, 15 μg of bovine serum albumin, 0.2 μM DTT, and 10 μM CPTwith or without SDD for 15 min at room temperature.

Reactions were stopped by the addition of 0.5% SDS, loading buffer (i.e.80% formamide, 10 μM NaOH, 1 μM Na₂EDTA, 0.1% xylencyanol, 0.1%bromphenol blue) in a proportion of (3:1) and the samples were resolvedin 16 or 20% acrylamide DNA sequencing gels containing 7 M urea.

Imaging and quantification of the cleavage products were performed usinga Typhoon Phosphorimager (Amersham Biosciences) as described inInhibition of Topoisomerase I cleavage activity by thiol-reactivecompounds. Montaudon, D.; Palle, K.; Rivory, L.; Robert, J.;Douat-Casassus, C.; Quideau S.; Bjornsti M.; Pourquier P; J. Biol. Chem.(2007), 282, 14403-14412.

Results

SDD used in four different concentrations: 1, 10, 100 and 1000 μM hasrevealed no Top 1 inhibitory activity compared with camptothecin.

Evaluation of the Effect of SDD on Catalytic Activity of TopoisomeraseII.

It is generally admitted that nuclear enzyme DNA topoisomerase II (TopoII) is the common target for a variety of anticancer drugs, includinganthracyclines, acridines, epipodophyllotoxines and ellipticines.Despite the fact that all these drugs induce formation and stabilizationof a ternary complex DNA-drug-Topo II, the domains and mechanisms ofspecific binds seems not to be the same for different chemical groups,which determine the pharmacologic characteristics of drugs such asefficacy and resistance.

Test for revelation of the possible inhibitory effect of SDD on Topo IIcatalytic activity has been undertaken by decatenation of a catenatedDNA substrate originating from Trypanosoma kinetoplasts (kDNA, TopoGen)into relaxed DNA forms.

Method.

The following standard reaction mixture contained 40 mM Tris/HCl pH 7.4,10 mM MgCl₂, 0.5 mM dithiothreitol, 1 mM ATP and 0.02 g/ml kDNA has beencarried out.

The reaction was initiated by the addition of 0.35 M NaCl nuclearextract and stopped after 20 min incubation at 37° C. by adding adenaturating solution containing 30% glycerol, 1% SDS, 0.5 mg/mlbromphenol blue.

The samples were then electrophoresed on a 1% agarose gel in 40 mMTris/acetic acid, pH 8.0 containing 2 mM EDTA and 0.1 μg/ml ethidiumbromide, for 45 min at 80V.

A positive control for decatenation was a decatenated kDNA markerobtained from TopoGen, containing a mixture of two species of relaxed

DNA, open circular DNA and covalently closed circular DNA. Catenated DNAdoes not enter the gel, while decatenated DNA circles do enter the geland migrate.

DNA was visualized under UV light and the various DNA forms werequantified by densitometric scanning using the device described above.

Etoposide was used as positive control, as it is described in thefollowing publication: Differential stabilisation oftopoisomerase-II-DNA cleavable complexes doxorubicine and etoposide indoxorubicin-resistant rat glioblastoma cells. Montaudon, D.; Pourquier,P.; Denois, F.; De Tinguy-Moreaud, E.; Lagarde, P.; Robert, J.; Eur., J.Biochem. (1997), 245, 307-315.

Results

SDD used in concentrations 1, 10, 100 and 1000 μM has caused relativelymoderate inhibition of Topo II activity compared to etoposide in doseindependent manner. Thus, there is insignificant inhibition of the TopoII activity, but, it does not depend on the concentrations of SDD.

Acute Toxicity Determination for SDD (LD₅₀)

The study has been performed to assess the acute systemic toxicity ofSDD in the mouse preliminary test.

The SDD was administered, by intravenous route:

-   -   in a first step to 1 female Swiss mouse at the single dose of 95        mg/kg b.w.,    -   in a second step to 1 female Swiss mouse at the single dose of        60 mg/kg b.w.,    -   in a third step to 1 female Swiss mouse at the single dose of 55        mg/kg b.w., and    -   in a fourth step to a group of 2 female Swiss mice at the single        dose of 50 mg/kg b.w.

It was noted the death of the animal treated at 95 mg/kg b.w.,immediately after the test item administration.

It was noted the death of the animal treated at 60 mg/kg b.w., 3 hoursafter the test item administration.

The mortality was preceded by a decrease of the spontaneous activityassociated with a decrease of Preyer's reflex, an absence of therighting reflex, a decrease of muscle tone and the eyes partly closed.

It was noted the death of the animal treated at 55 mg/kg b.w., 24 hoursafter the test item administration. The mortality was preceded by adecrease of the spontaneous activity associated with a decrease of therighting reflex.

No mortality occurred in the animals treated at 50 mg/kg b.w.

It was registered, 1 hour after the test item administration, a decreaseof the spontaneous activity (2/2) (i.e. 2 mice out of 2 mice) associatedwith a decrease of the righting reflex (1/2). The animals recovered anormal activity the second day of the test.

It was noted an oedema at the level of the tails of the mice between thefirst day and the fourteenth day.

The body weight evolution of the animals remained normal throughout thestudy. The macroscopical examination of the animals treated at 50 mg/kgat the end of the study did not reveal any treatment-related changes.

Main Test

The SDD was administered by intravenous route at the dose of 50 mg/kgbody weight to a group of 5 female Swiss mice. The experimental protocolwas established from the International standard NF EN ISO 10993-11concerning biological evaluation of medical devices: “Tests for systemictoxicity”.

No mortality occurred during the study. It was registered in the treatedanimals, 1 hour after the test item administration, a decrease of thespontaneous activity (5/5) associated with a decrease of the rightingreflex (2/5) and the eyes partly closed (2/5). The animals recoverednormal activity the 2nd day of the test.

It was noted an oedema at the level of the tails of mice between thefirst day and the fourteenth day. The body weight evolution of animalsremained normal throughout the study. The macroscopical examination ofanimals treated at 50 mg/kg at the end of the study revealed onlynecrosis at the level of the tail (3/5).

CONCLUSION

In conclusion, the mouse LD₅₀ for SDD is found above 50 mg/kg of bodyweight, when administered by intravenous route.

1. Compound of the general structure (I)

wherein n is an integer comprised between 0 and 10, preferably between 3and
 10. 2. Compound according to claim 1 wherein n is comprised between4 and
 8. 3. Compound according to claim 1 wherein n is equal to 5 or 6or
 7. 4. Compound according to claim 1 which is thebis[O-(14-benzoylaconine-8-yl)]-suberate.
 5. Method for the productionof a compound having the general structure (I), wherein said methodcomprises the following steps: a) stirring aconitine with a dicarboxylicacid at a temperature equal or lower than 80° C. in a solvent such DMF,b) agitating the obtained mixture at room temperature such that thereaction of transesterification is carried out, c) evaporating thesolvent under reduced pressure, d) purification of the oily residueobtained at the end of the step c) to isolate the said compound (I). 6.Method according to claim 5 wherein the dicarboxylic acid has a numberof carbon atoms comprised between 2 and 12, preferably between 5 and 12,more preferably between 6 and
 10. 7. Method according to claim 6 whereinthe dicarboxylic acid is selected from the group consisting of pimelicacid and suberic acid and azelaic acid.
 8. A pharmaceutical compositioncomprising a compound as defined in claim
 1. 9. A pharmaceuticalcomposition according to claim 8 which further comprises a suitable,pharmaceutically acceptable diluent or carrier substance.
 10. Use of thecompound according to claim 1 for the fabrication of a medicament. 11.Use according to claim 10 in which the medicament is used for thetreatment of tumour diseases.
 12. Use according to claim 11 in which themedicament is used for the treatment of colon or lung or breast cancer.